Code Authors: Mark E. Pepin, MD, PhD, MS Contact:
Institution: Brigham and Women’s Hospital | Broad Institute of Harvard and MIT
Location: Boston, MA

Data Pre-Processing

library(Seurat)
# Upload data provided
hdat <- readRDS(file = "../1_Input/snRNA_Human_ControlDissection_integrated_seurat_v2.rds")
# Initialize the Seurat object with the raw (non-normalized data).
hdat[["percent.mt"]] <- PercentageFeatureSet(hdat, pattern = "^MT-")
hdat <- subset(hdat, subset = nFeature_RNA > 200 & nFeature_RNA < 2500 & percent.mt < 5)
hdat <- NormalizeData(hdat, normalization.method = "LogNormalize", scale.factor = 10000)
hdat <- FindVariableFeatures(hdat, selection.method = "vst", nfeatures = 10000)
all.genes <- rownames(hdat)
hdat <- ScaleData(hdat, features = all.genes)
hdat[["Celltype_annotation_v2"]]$Celltype_annotation_v2 <- factor(hdat[["Celltype_annotation_v2"]]$Celltype_annotation_v2, c("VSMC1", "VSMC2", "Myofibroblast", "Fibroblast1", "Fibroblast2", "EC1", "EC2", "Macrophage", "NKT Cells", "Neuronal"))
##
TAA_list<-SplitObject(hdat, split.by = "Disease") # split dataset into a list of two seruat objects
# normalize and identify variable features for each dataset independently
TAA.list <- lapply(X = TAA_list, FUN = function(x) {
    x <- NormalizeData(x)
    x <- FindVariableFeatures(x, selection.method = "vst", nfeatures = 10000)
})
# select features that are repeatedly variable across datasets for integration
features <- SelectIntegrationFeatures(object.list = TAA.list)
# Preserve cell type markers in the anchoring dataset
VSMC1_genes<-c("PKD1", "COL6A2", "PDLIM7", "FLNA", "SMTN")
VSMC2_genes<-c("MYH11", "ACTA2", "ITGA8", "PRKG1", "CRISPLD1")
Fibroblast1_genes<-c("ABCA10", "C3", "ADGRD1", "FBLN1", "DCN")
Fibroblast2_genes<-c( "NFASC",  "SAMD5","UACA","PRSS23") #  "TEX41",
Fibromyocyte_genes<-c("DGKG", "ADAMTS1", "RGS6", "TNC", "GRIP2", "ANGPT2")
EC1_genes<-c("DIPK2B", "ARHGEF15", "STC1", "FLT1") #, "NOTCH4"
EC2_genes<-c("VWF", "BMPER", "BMX", "NOS1")
NKT_genes<-c("SKAP1", "RIPOR2", "RBPJ", "FYN", "ITGAL", "CD96")
Macrophage_genes<-c("MRC1", "LGMN", "F13A1", "RBM47")
Dendritic_genes<-c("ITGAX", "S100A9", "CSF3R", "CXCL8")
features<-union(features, union(VSMC1_genes,VSMC2_genes))
#   VSMC1_genes, union(
#     VSMC2_genes, union(
#       Fibroblast1_genes, union(
#         Fibroblast2_genes, union(
#           Fibromyocyte_genes, union(
#             EC1_genes, union(
#               EC2_genes, union(
#                 NKT_genes, union(Macrophage_genes, Dendritic_genes)
#                 )
#               )
#             )
#           )
#         )
#       )
#   )
# )
# )
# Identify "anchors" (cells that are unchanged across datasets)
TAA.anchors <- FindIntegrationAnchors(object.list = TAA.list, anchor.features = features)
# this command creates an 'integrated' data assay
TAA.combined <- IntegrateData(anchorset = TAA.anchors)
# Specify the matrix used in the downstream analysis
DefaultAssay(TAA.combined) <- "integrated"
# Run the standard workflow for visualization and clustering
TAA.combined <- ScaleData(TAA.combined, verbose = FALSE)
TAA.combined <- RunPCA(TAA.combined, npcs = 30, verbose = FALSE)
TAA.combined <- RunUMAP(TAA.combined, dims = 1:30)
TAA.combined <- RunTSNE(TAA.combined)
TAA.combined <- FindNeighbors(TAA.combined, dims = 1:30)
TAA.combined <- FindClusters(TAA.combined, resolution = 0.4)
## Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
## 
## Number of nodes: 9278
## Number of edges: 381125
## 
## Running Louvain algorithm...
## Maximum modularity in 10 random starts: 0.8951
## Number of communities: 11
## Elapsed time: 1 seconds
saveRDS(TAA.combined, file = "TAA_FFPE_snRNA.rds")
#
plot1 <- VariableFeaturePlot(hdat)
top10 <- head(VariableFeatures(hdat), 10)
plot2 <- LabelPoints(plot = plot1, points = c(top10), repel = TRUE)
pdf(file = "../2_Output/Figure_1/FFPE_Variable_genes.pdf")
plot2
dev.off()
## quartz_off_screen 
##                 2

Figure 1

Clustering

# Clustering
umap_disease <- DimPlot(hdat, reduction = "umap", group.by = "Disease")
umap_clusters <- DimPlot(TAA.combined, reduction = "umap", label = TRUE, repel = TRUE)
pca_disease <- DimPlot(TAA.combined, reduction = "pca", group.by = "Disease")
pca_clusters <- DimPlot(TAA.combined, reduction = "pca", label = TRUE, repel = TRUE)
tsne_disease <- DimPlot(TAA.combined, reduction = "tsne", group.by = "Disease")
tsne_clusters <- DimPlot(TAA.combined, reduction = "tsne", label = TRUE, repel = TRUE)
pdf("../2_Output/Supplemental_Figures/Clustering_Comparison.pdf", width = 11, height = 8)
umap_disease+pca_disease+tsne_disease+umap_clusters+pca_clusters+tsne_clusters
dev.off()

# Add tracings around the points
library(ggplot2)
library(ggtrace)
library(ggthemes)
library(ggrepel)
library(dplyr)
# UMAP
umap_data <- as.data.frame(TAA.combined@reductions$umap@cell.embeddings) # Create a data.frame from the UMAP (to be used in ggplot)
# Disease Clustering
umap_data$Disease <- TAA.combined$Disease
umap_data$Disease <- factor(umap_data$Disease, levels = c("Control", "Dissection"))
umap_disease <- ggplot(umap_data, aes(x = UMAP_1, y = UMAP_2, fill = Disease)) +
  geom_point_trace(alpha = .25, stroke = .5, size = 1, color = "black")  +
  theme_classic() +
  theme(text = element_text(size = 14))
umap_disease

### UMAP Clustering
umap_data$Cluster <- TAA.combined$seurat_clusters # Create a variable based on UMAP clusters (Seurat)
cluster_centers <- aggregate(cbind(UMAP_1, UMAP_2) ~ Cluster, umap_data, mean) # Label Clusters
umap_clusters <- ggplot(umap_data, aes(x = UMAP_1, y = UMAP_2, fill = Cluster)) +
  geom_point_trace(alpha = .25, stroke = .5, size = 1, color = "black")  +
  theme_classic() + 
  geom_text_repel(data = cluster_centers, aes(label = Cluster), size = 6) +
  theme(legend.position = "none", text = element_text(size = 14))
umap_clusters

pdf(file = "../2_Output/Supplemental_Figures/UMAP_overlay.pdf", width = 11.5, height = 5, bg = "transparent")
umap_disease + umap_clusters
dev.off()

#Figure 1C -  Proportional Graph
library(dittoSeq)
dittoBarPlot(
    object = hdat,
    var = "Celltype_annotation_v2",
    group.by = "Disease")

dittoBarPlot(
    object = hdat,
    var = "Celltype_annotation_v2",
    group.by = "Sample_name",
    split.by = "Disease")

######################################################
############## Dimensionality
#####################################################
# to control for any confounders, we can add the 'vars.to.regress' parameter, including any variable in the metadata <- ScaleData(pbmc, vars.to.regress = "percent.mt")
TAA.combined <- RunPCA(TAA.combined, features = VariableFeatures(object = TAA.combined))
# Visualize the features/genes
VizDimLoadings(TAA.combined, dims = 1:5, reduction = "pca")

DimPlot(TAA.combined, reduction = "umap")

pdf(file = "../2_Output/Supplemental_Figures/PC_Heatmaps.pdf")
DimHeatmap(TAA.combined, dims = 1:15, cells = 500, balanced = TRUE)
dev.off()
# Determine the number of dimensions represented by the data
TAA.combined <- JackStraw(TAA.combined, num.replicate = 100)
TAA.combined <- ScoreJackStraw(TAA.combined, dims = 1:20)
pdf(file = "../2_Output/Supplemental_Figures/JackStraw.pdf")
JackStrawPlot(TAA.combined, dims = 1:20)
dev.off()
pdf(file = "../2_Output/Supplemental_Figures/ElbowPlot.pdf")
ElbowPlot(TAA.combined)
dev.off()
######################################################
############## Cluster Annotation
#####################################################
library(ggplot2)
library(dplyr)
library(Seurat)
# TAA.combined<-readRDS(file = "TAA.ALL_snRNA.rds") # contains ACTA2, normal, and aortic aneurism (Cheu et. al)
# Find all gene markers
DEGs_Clusters<-FindAllMarkers(TAA.combined, assay = "RNA")
write.csv(DEGs_Clusters, "../2_Output/Supplemental_Figures/DEGs_Clusters.csv")
#Identify Clusters corresponding with known gene markers:
pdf(file = "../2_Output/Supplemental_Figures/UMAP_split.pdf", height = 4, width = 7)
DimPlot(TAA.combined,  label = T, split.by = "Disease") + NoLegend()
dev.off()
pdf(file = "../2_Output/Supplemental_Figures/UMAP_samples.pdf", height = 4, width = 11)
DimPlot(TAA.combined,  label = T, split.by = "Sample_name") + NoLegend()
dev.off()
# Create a dot-bplot of the top 5 markers for each 
library(scCustomize)
paletteLength <- 100
myColor <- colorRampPalette(c("dodgerblue4", "white", "coral2"))(paletteLength)
top5_markers <- Extract_Top_Markers(marker_dataframe = DEGs_Clusters, num_genes = 10, named_vector = FALSE,
    make_unique = TRUE)
pdf(file = "../2_Output/Supplemental_Figures/Dot.plot_top5markers.pdf", height = 10, width = 7)
Clustered_DotPlot(seurat_object = TAA.combined, features = top5_markers, k = 10, colors_use_exp = myColor)
dev.off()
saveRDS(TAA.combined, file = "TAA_FFPE_snRNA.rds")

Cell-type Annotation

library(Seurat)
library(ggrepel)
TAA.combined<-readRDS(file = "TAA_FFPE_snRNA.rds")
#Identify Clusters corresponding with known gene markers:
VSMC1_genes<-c("PKD1", "COL6A2", "PDLIM7", "FLNA", "SMTN")
VSMC2_genes<-c("MYH11", "ACTA2", "ITGA8", "PRKG1", "CRISPLD1")
Fibroblast1_genes<-c("ABCA10", "C3", "ADGRD1", "FBLN1", "DCN")
Fibroblast2_genes<-c("NFASC",  "SAMD5", "PRSS23") #"UACA","TEX41",
Fibromyocyte_genes<-c("ADAMTS1", "RGS6", "TNC") # , "ANGPT2", "DGKG", "GRIP2"
EC1_genes<-c("DIPK2B", "ARHGEF15", "STC1", "FLT1") # , "NOTCH4"
EC2_genes<-c("VWF", "BMPER", "BMX", "NOS1")
NKT_genes<-c("SKAP1", "RIPOR2", "ITGAL", "CD96") #  "RBPJ", "FYN",
Macrophage_genes<-c("MRC1", "LGMN", "F13A1", "RBM47") #
Dendritic_genes<-c("ITGAX", "S100A9", "CSF3R", "CXCL8") #
# Plot density function
library(Nebulosa)
VSMC1_density<-plot_density(TAA.combined, VSMC1_genes, reduction = "umap", joint = TRUE, combine = FALSE, pal = "magma")
VSMC2_density<-plot_density(TAA.combined, VSMC2_genes, reduction = "umap", joint = TRUE, combine = FALSE, pal = "magma")
Fibroblast1_density<-plot_density(TAA.combined, Fibroblast1_genes, reduction = "umap", joint = TRUE, combine = FALSE, pal = "magma")
Fibroblast2_density<-plot_density(TAA.combined, Fibroblast2_genes, reduction = "umap", joint = TRUE, combine = FALSE, pal = "magma")
Fibromyocyte_density<-plot_density(TAA.combined, Fibromyocyte_genes, reduction = "umap", joint = TRUE, combine = FALSE, pal = "magma")
EC1_density<-plot_density(TAA.combined, EC1_genes, reduction = "umap", joint = TRUE, combine = FALSE, pal = "magma")
EC2_density<-plot_density(TAA.combined, EC2_genes, reduction = "umap", joint = TRUE, combine = FALSE, pal = "magma")
Macrophage_density<-plot_density(TAA.combined, Macrophage_genes, reduction = "umap", joint = TRUE, combine = FALSE, pal = "magma")
NKT_density<-plot_density(TAA.combined, NKT_genes, reduction = "umap", joint = TRUE, combine = FALSE, pal = "magma")
pdf(file = "../2_Output/Supplemental_Figures/Density_plots.pdf")
VSMC1_density
VSMC2_density
Fibroblast1_density
Fibroblast2_density
EC1_density
EC2_density
Macrophage_density
NKT_density
Fibromyocyte_density
dev.off()

# Overlay these gene markers onto the UMAP to identify clusters
pdf(file = "../2_Output/CellType_FeaturePlots.pdf")
FeaturePlot(TAA.combined, reduction = "umap", label = T, features = Fibroblast1_genes)
FeaturePlot(TAA.combined, reduction = "umap", label = T, features = Fibroblast2_genes)
FeaturePlot(TAA.combined, reduction = "umap", label = T, features = EC1_genes)
FeaturePlot(TAA.combined, reduction = "umap", label = T, features = EC2_genes)
FeaturePlot(TAA.combined, reduction = "umap", label = T, features = NKT_genes)
FeaturePlot(TAA.combined, reduction = "umap", label = T, features = Macrophage_genes)
FeaturePlot(TAA.combined, reduction = "umap", label = T, features = VSMC1_genes)
FeaturePlot(TAA.combined, reduction = "umap", label = T, features = VSMC2_genes)
# FeaturePlot(TAA.combined, reduction = "umap", label = T, features = Dendritic_genes)
dev.off()
#Create a figure of cell-type specific markers overlying the UMAP
pdf(file = "../2_Output/Supplemental_Figures/CellType_Differentiation.pdf")
plot_density(TAA.combined, c("ACTA2", "FBLN1", "F13A1", "VWF", "STC1", "NFASC", "ITGAL", "ITGAX"), reduction = "umap", joint = TRUE, combine = FALSE, pal = "magma")
dev.off()
# Change the identity of the clusters to cell types
Idents(TAA.combined) <- "Celltype_annotation_v2"
# Create UMAP with annotated cell-types
UMAP_CellTypes<-DimPlot(TAA.combined,  label = T) + NoLegend()
UMAP_CellTypes

#######
umap_data <- as.data.frame(TAA.combined@reductions$umap@cell.embeddings) # Create a data.frame from the
umap_data$CellType<-TAA.combined@active.ident
cluster_centers <- aggregate(cbind(UMAP_1, UMAP_2) ~ CellType, umap_data, mean) # Label Clusters
umap_celltype <- ggplot(umap_data, aes(x = UMAP_1, y = UMAP_2, fill = CellType)) +
  # geom_point() +
  # scale_fill_brewer(palette="Set2") + 
  geom_point_trace(alpha = .25, stroke = .5, size = 1, color = "black")  +
  theme_classic() + 
  geom_text_repel(data = cluster_centers, aes(label = CellType), size = 5) +
  theme(legend.position = "none", text = element_text(size = 12))
## Fully Annotated Merged UMAP
pdf(file = "../2_Output/UMAP_Disease.Overlay.pdf", height = 5, width = 5)
umap_celltype
dev.off()

pdf(file = "../2_Output/Supplemental_Figures/UMAP_All.three_Labelled.pdf", height = 8, width = 16)
umap_disease+theme(legend.position = "topright", text = element_text(size = 14)) + 
umap_clusters+umap_celltype
dev.off()

## Use ridgeplots to identify bimodal gene marker distributions (enriched clusters)
pdf(file = "../2_Output/Celltype_RidgePlots.pdf", height = 10, width = 15)
RidgePlot(TAA.combined,features = Fibroblast1_genes, ncol = 2)
RidgePlot(TAA.combined, features = Fibroblast2_genes, ncol = 2)
RidgePlot(TAA.combined, features = EC1_genes, ncol = 2)
RidgePlot(TAA.combined, features = EC2_genes, ncol = 2)
RidgePlot(TAA.combined, features = NKT_genes, ncol = 2)
RidgePlot(TAA.combined, features = Macrophage_genes, ncol = 2)
RidgePlot(TAA.combined, features = VSMC1_genes, ncol = 2)
RidgePlot(TAA.combined, features = VSMC2_genes, ncol = 2)
RidgePlot(TAA.combined, features = Dendritic_genes, ncol = 2)
dev.off()
# Differential Expression
# Export DEGs using cell-type clusters
DEGs_CellTypes<-FindAllMarkers(TAA.combined)
write.csv(DEGs_CellTypes, "../2_Output/DEGs_Clusters.csv")
# Create a dot-bplot of the top 5 markers for each 
library(scCustomize)
paletteLength <- 100
myColor <- colorRampPalette(c("dodgerblue4", "white", "coral2"))(paletteLength)
top5_markers <- Extract_Top_Markers(marker_dataframe = DEGs_CellTypes, num_genes = 5, named_vector = FALSE,
    make_unique = TRUE)
pdf(file = "../2_Output/Dot.plot_top5markers.pdf", height = 10, width = 7)
Clustered_DotPlot(seurat_object = TAA.combined, features = top5_markers, k = 10, colors_use_exp = myColor)
dev.off()
# Heatmap of Clusters
DEGs_CellTypes %>%
    group_by(cluster) %>%
    top_n(n = 10, wt = avg_log2FC) -> top10
pdf(file = "../2_Output/Celltype_Heatmap.pdf", height = 10, width = 15)
DoHeatmap(TAA.combined, features = top10$gene, size = 3, disp.min = -2, disp.max = 2) + scale_fill_gradientn(colors = c("dodgerblue4", "white", "coral2")) + labs(title = "Heatmap of Top-10 most variable genes within each cluster")
dev.off()
# Probability distribution of specific genes across all clusters/cell types
VlnPlot(TAA.combined, features = c("VWF", "HDAC9", "ACTA2"))

#
DotPlot(TAA.combined, features = c(VSMC1_genes, VSMC2_genes, EC1_genes, EC2_genes, Fibroblast1_genes, Fibroblast2_genes, Fibromyocyte_genes, Macrophage_genes, NKT_genes)) + RotatedAxis()

saveRDS(TAA.combined, file = "TAA_FFPE_snRNA.rds")

Figure 2: VSMC-Specific Analysis

VSMC Phenotypic Comparison

library(Seurat)
library(dplyr)
library(ggtrace)
library(ggplot2)
library(ggrepel)
# Add tracings around the points
library(ggplot2)
library(ggtrace)
library(ggthemes)
library(ggrepel)
library(dplyr)
hdat_vsmc <- subset(TAA.combined, Celltype_annotation_v2==c("VSMC1", "VSMC2"))
UMAP_VSMC<-DimPlot(hdat_vsmc,  label = T) + NoLegend()
# Figure 1A - UMAP of VSMCs only
pdf("../2_Output/Figure_2/Fig2A_UMAP_VSMC.pdf", height = 3.5, width = 3.5)
UMAP_VSMC
dev.off()

library(Nebulosa)
VSMC1_density<-plot_density(hdat_vsmc, VSMC1_genes, reduction = "umap", joint = TRUE, combine = FALSE, pal = "magma")
VSMC2_density<-plot_density(hdat_vsmc, VSMC2_genes, reduction = "umap", joint = TRUE, combine = FALSE, pal = "magma")
pdf(file = "../2_Output/Figure_2/Fig2A_VSMC_Density_plots.pdf", height = 3.5, width = 3.5)
VSMC1_density
VSMC2_density
dev.off()

# Visualization
p1 <- DimPlot(hdat_vsmc, reduction = "umap", group.by = "Disease")
p2 <- DimPlot(hdat_vsmc, reduction = "umap", label = TRUE, repel = TRUE)
p1 + p2

umap_data <- as.data.frame(hdat_vsmc@reductions$umap@cell.embeddings) # Create a data.frame from the UMAP (to be used in ggplot)
# Disease Clustering
umap_data$Disease <- hdat_vsmc$Disease
umap_data$Disease <- factor(umap_data$Disease, levels = c("Control", "Dissection"))
p1 <- ggplot(umap_data, aes(x = UMAP_1, y = UMAP_2, fill = Disease)) +
  geom_point_trace(alpha = .25, stroke = .5, size = 1, color = "black")  +
  theme_classic() +
  theme(text = element_text(size = 14))
p1

umap_TAA<-umap_data %>% filter(Disease=="Control")
p_TAA <- ggplot(umap_TAA, aes(x = UMAP_1, y = UMAP_2, fill = Disease)) +
  geom_point_trace(alpha = .25, stroke = .5, size = 1, color = "black")  +
  theme_classic() +
  theme(text = element_text(size = 14))
p_TAA

umap_ACTA<-umap_data %>% filter(Disease=="Dissection")
p_ACTA <- ggplot(umap_ACTA, aes(x = UMAP_1, y = UMAP_2, fill = Disease)) +
  geom_point_trace(alpha = .25, stroke = .5, size = 1, color = "black")  +
  theme_classic() +
  theme(text = element_text(size = 14))
p1 + p_TAA + p_ACTA

# Create the UMAP of cell-type specific clusters
UMAP_CellTypes<-DimPlot(hdat_vsmc, label = T) + NoLegend()
UMAP_CellTypes

umap_data <- as.data.frame(hdat_vsmc@reductions$umap@cell.embeddings) # Create a data.frame from the
umap_data$Cluster<-hdat_vsmc@active.ident
cluster_centers <- aggregate(cbind(UMAP_1, UMAP_2) ~ Cluster, umap_data, mean) # Label Clusters
p3 <- ggplot(umap_data, aes(x = UMAP_1, y = UMAP_2, fill = Cluster)) +
  # geom_point() +
  # scale_fill_brewer(palette="Set2") + 
  geom_point_trace(alpha = .25, stroke = .5, size = 1, color = "black")  +
  theme_classic() + 
  geom_text_repel(data = cluster_centers, aes(label = Cluster), size = 6) +
  theme(legend.position = "none", text = element_text(size = 14))
p3

pdf(file = "../2_Output/Figure_2/VSMC_UMAP_CellTypes.pdf", width = 11.5, height = 5, bg = "transparent")
p1+theme(legend.position = "top", text = element_text(size = 14)) + p3
dev.off()

# Differential Expression between VSMCs
VSMC1.markers <- FindMarkers(hdat_vsmc, ident.1 = "VSMC1", min.pct = 0.25, logfc.threshold = 0.25)
VSMC2.markers <- FindMarkers(hdat_vsmc, ident.1 = "VSMC2", min.pct = 0.25, logfc.threshold = 0.25)

#Volcano Plot
# Load packages
library(dplyr)
library(ggplot2)
library(ggrepel)
library(openxlsx)
# Read data from the web
options(ggrepel.max.overlaps = Inf)
results = mutate(VSMC2.markers, minuslogpvalue = -log(p_val), log2FC=avg_log2FC)
results<-results %>% filter(p_val!=0)
results$gene_name<-rownames(results)
results <- results %>% 
  mutate(., sig=ifelse(p_val<0.05 & log2FC>.5, 
                       "P < 0.05 and Fold-Change > 0.5", 
                       ifelse(p_val<0.05 & log2FC< 0-0.5,
                              "P < 0.05 and Fold-Change < -0.5", 
                              "Not Sig")
                       )
         )
results$sig<-factor(results$sig, 
levels = c("P < 0.05 and Fold-Change < -0.5",
  "Not Sig",
  "P < 0.05 and Fold-Change > 0.5")
  )
max(results$minuslogpvalue, na.rm = TRUE)
max(results$log2FC, na.rm = TRUE)
min(results$log2FC, na.rm = TRUE)
p = ggplot(results, aes(log2FC, minuslogpvalue)) + 
  theme_classic() +
  theme(axis.line = element_blank(),
        axis.ticks = element_blank()
  ) +
  geom_point(aes(fill=sig, size = minuslogpvalue),
             colour="black",
             shape=21,
             stroke = 0,
             alpha = .9) +
  geom_vline(xintercept=0.5, size=.5, linetype="dashed") +
  geom_vline(xintercept=-0.5, size=0.5, linetype="dashed") +
  geom_hline(yintercept=0-log(0.05), size=.5, linetype="dashed") +
  labs(x=expression(Log[2](Fold-Change)), y=expression(-Log[10](P-value))) + 
  xlim(min(results$log2FC, na.rm = TRUE),max(results$log2FC, na.rm = TRUE)) + 
  ylim(0, max(results$minuslogpvalue, na.rm = TRUE)) + geom_hline(yintercept = 0, size = 1) + 
  geom_vline(xintercept=0, size=1) +
  scale_fill_manual(values=c("darkcyan", "darkgray", "coral2")) +
  scale_size_continuous(range = c(.1, 3))

  p+
  geom_text_repel(data=top_n(filter(results, log2FC< -0.5), 10, minuslogpvalue), aes(label=gene_name)) +
  geom_text_repel(data=top_n(filter(results, log2FC>0.5), 10, minuslogpvalue), aes(label=gene_name)) +
  theme(text = element_text(size=20))

##
pdf(file = paste0("../2_Output/Figure_2/VSMC_1.vs.2_VolcanoPlot.pdf"), height = 5, width = 5)
  p+
  geom_text_repel(data=top_n(filter(results, log2FC< -0.5), 10, minuslogpvalue), aes(label=gene_name)) +
  geom_text_repel(data=top_n(filter(results, log2FC>0.5), 10, minuslogpvalue), aes(label=gene_name)) +
    theme(text = element_text(size=14), legend.position="none")
dev.off()
# Create a file of differentially expressed genes between VSMC1 and VSMC2
openxlsx::write.xlsx(VSMC1.markers, file = "../2_Output/Figure_2/VSMC_1v2_DEGs.xlsx", rowNames=T)

VSMC_type_DEs <- results$gene_name[1:100]
##
plots_VSMC2 <- VlnPlot(hdat_vsmc, features = c("PALLD", "MAP2", "PRKG1"), group.by = "Celltype_annotation_v2",
    pt.size = 0) & theme(legend.position = 'none', axis.title.x = element_blank())
plots_VSMC1 <- VlnPlot(hdat_vsmc, features = c("B2M", "TNFAIP2", "STAB1"), group.by = "Celltype_annotation_v2",
    pt.size = 0) & theme(legend.position = 'none', axis.title.x = element_blank())

pdf(file = "../2_Output/Figure_2/VSMC2_Top.Violins.pdf", height = 3, width = 6)
plots_VSMC2
dev.off()
pdf(file = "../2_Output/Figure_2/VSMC1_Top.Violins.pdf", height = 3, width = 6)
plots_VSMC1
dev.off()

##################
DoHeatmap(hdat_vsmc, features = VSMC_type_DEs, group.by = "Celltype_annotation_v2",size = 3, disp.min = -2, disp.max = 2) + scale_fill_gradient2(low = "dodgerblue4", mid = "white", high = "coral2",midpoint = 0)

##################
pdf(file = "../2_Output/Figure_2/DotPlot_VSMC_1.v.2.pdf", height = 10, width = 5)
Clustered_DotPlot(seurat_object = hdat_vsmc, features = VSMC_type_DEs, group.by = "Celltype_annotation_v2", x_lab_rotate=F, k = 2)
dev.off()

VSMC Trajectory Analysis (Monocle 3)

library(monocle3)
library(Seurat)
library(SeuratWrappers)
library(dplyr)
library(ggtrace)
Merged_scaled<-hdat_vsmc
Merged_scaled@active.assay = "RNA"
cds<-SeuratWrappers::as.cell_data_set(Merged_scaled)
cds <- preprocess_cds(cds, num_dim = 100)
cds <- estimate_size_factors(cds)
# Include gene names (not done by default by the seurat conversion)
rowData(cds)$gene_name <- rownames(cds)
rowData(cds)$gene_short_name <- rowData(cds)$gene_name
#  Run Monocle
cds <- cluster_cells(cds) # This step creates "partitions" that are used in the trajectory inference
plot_cells(cds, show_trajectory_graph = FALSE, color_cells_by = "partition") # this shows the partitions overlain on the UMAP

cds <- learn_graph(cds, use_partition = TRUE) # creating trajectory inference within each partition
# cds <- order_cells(cds) # Used when setting the nodes; if already known, use the next line
root_cell <- "TACTCGCTCCCTGACT-1-1" # names(which(cds@principal_graph_aux$UMAP$pseudotime==0))
cds<-order_cells(cds, root_cells = root_cell)

# Plot the pseudotime on UMAP
plot_cells(cds, 
           color_cells_by = "pseudotime",
           label_branch_points = FALSE,
           label_leaves = FALSE)

pdf(file = "../2_Output/Figure_2/VSMC_UMAP_Trajectory_Partition.pdf", height = 3, width = 3)
plot_cells(cds,
           color_cells_by = "partition",
           graph_label_size = 1,
           cell_size = .5,
           label_roots = F,
           label_branch_points = FALSE,
           label_leaves = FALSE)
dev.off()
pdf(file = "../2_Output/Figure_2/VSMC_UMAP_Trajectory_Pseudotime.pdf", height = 3, width = 4)
plot_cells(cds,
           color_cells_by = "pseudotime",
           graph_label_size = 1,
           cell_size = .7,
           label_branch_points = FALSE,
           label_leaves = F)
dev.off()

# Identify pseudotime
modulated_genes <- graph_test(cds, neighbor_graph="principal_graph", cores=8) # Identify differentially-expressed genes with pseudotime
modulated_genes <- na.omit(modulated_genes) # remove NA's
modulated_genes <- modulated_genes[modulated_genes$q_value < 0.05 & modulated_genes$status =="OK", ] # filter cds results down
modulated_genes <- modulated_genes[order(-modulated_genes$morans_test_statistic), ] # order by moran's test
#### Create a heatmap of genes with similar pseudotime kinetics
genes <- row.names(subset(modulated_genes, q_value < 0.05 & morans_I > 0.2))
openxlsx::write.xlsx(modulated_genes, "../2_Output/Figure_2/Pseudotime_DEGs.xlsx")

library(ComplexHeatmap)
library(ggplot2)
library(dplyr)
library(RColorBrewer)
library(circlize)
library(monocle3)
pt.matrix <- exprs(cds)[match(genes,rownames(rowData(cds))),order(pseudotime(cds))]
#Can also use "normalized_counts" instead of "exprs" to use various normalization methods, for example:
#normalized_counts(cds, norm_method = "log")
pt.matrix <- t(apply(pt.matrix,1,function(x){smooth.spline(x,df=3)$y})) # Create a spline that smooths the pseudotime-based expression along the first 3 degrees of freedom.
pt.matrix <- t(apply(pt.matrix,1,function(x){(x-mean(x))/sd(x)}))
rownames(pt.matrix) <- genes;

#Ward.D2 Hierarchical Clustering
hthc <- Heatmap(
  pt.matrix,
  name                         = "z-score",
  col                          = colorRamp2(seq(from=-2,to=2,length=11),rev(brewer.pal(11, "Spectral"))),
  show_row_names               = TRUE,
  show_column_names            = FALSE,
  row_names_gp                 = gpar(fontsize = 6),
  clustering_method_rows = "ward.D2",
  clustering_method_columns = "ward.D2",
  row_title_rot                = 0,
  cluster_rows                 = TRUE,
  cluster_row_slices           = FALSE,
  cluster_columns              = FALSE)
pdf(file = "../2_Output/Figure_2/VSMC_Pseudotime.Heatmap.pdf", height = 4, width = 3)
print(hthc)
dev.off()

# Examine specific genes within the module(s) of interest (appears to emrich to metabolic genes)
library("viridis")
library(ggplot2)
GENES <- c("AEBP1", "LAMA2", "HDAC9", "ACTA2", "EXOC4", "PDE3A") #Genes selected based on visible trends
p1 <- plot_cells(cds, 
           genes = GENES[1:2],
           label_cell_groups = F,
           label_roots = F,
           label_branch_points = F,
           label_leaves = F,
           show_trajectory_graph = F,
           min_expr = 1,
           alpha = 0.8,
           trajectory_graph_color = "grey28",
           ) +
  # geom_point_trace(alpha = .25, stroke = .5, size = 1, color = "black")  +
  theme(
  axis.text = element_text(size = 6),    # Adjust the size as needed
  axis.title = element_text(size = 8),  # Adjust the size as needed
  legend.text = element_text(size = 4),  # Adjust the size as needed
  legend.title = element_text(size = 6),
  legend.key.size = unit(2, 'mm')) +
  scale_colour_viridis_c(option = "inferno")
p2 <- plot_cells(cds, 
           genes = GENES[3:4],
           label_cell_groups = F,
           label_roots = F,
           label_branch_points = F,
           label_leaves = F,
           show_trajectory_graph = F,
           min_expr = 1,
           alpha = 0.8,
           trajectory_graph_color = "grey28",
           ) +
  # geom_point_trace(alpha = .25, stroke = .5, size = 1, color = "black")  +
  theme(
  axis.text = element_text(size = 6),    # Adjust the size as needed
  axis.title = element_text(size = 8),  # Adjust the size as needed
  legend.text = element_text(size = 4),  # Adjust the size as needed
  legend.title = element_text(size = 6),
  legend.key.size = unit(2, 'mm')) +
  scale_colour_viridis_c(option = "inferno")
p3 <- plot_cells(cds, 
           genes = GENES[5:6],
           label_cell_groups = F,
           label_roots = F,
           label_branch_points = F,
           label_leaves = F,
           show_trajectory_graph = F,
           min_expr = 1,
           alpha = 0.8,
           trajectory_graph_color = "grey28",
           ) +
  # geom_point_trace(alpha = .25, stroke = .5, size = 1, color = "black")  +
  theme(
  axis.text = element_text(size = 6),    # Adjust the size as needed
  axis.title = element_text(size = 8),  # Adjust the size as needed
  legend.text = element_text(size = 4),  # Adjust the size as needed
  legend.title = element_text(size = 6),
  legend.key.size = unit(2, 'mm')) +
  scale_colour_viridis_c(option = "inferno")
pdf(file="../2_Output/Figure_2/UMAP_Pseudotime_Genes.pdf", height = 4, width = 3)
ggarrange(p1, p2,p3, ncol=1, nrow=3, common.legend = TRUE, legend="right")
dev.off()
p1+p2+p3

# Plot genes according to "pseudotime"
lineage_cds <- cds[rowData(cds)$gene_short_name %in% GENES, ] #colData(cds)$cell_type %in% c("VSMC")
lineage_cds<-order_cells(lineage_cds, root_cells = root_cell)
pdf(file="../2_Output/Figure_2/Pseudotime_curves.pdf", height = 7, width = 3)
monocle3::plot_genes_in_pseudotime(lineage_cds,
                         color_cells_by="ident",
                         min_expr=0)
dev.off()

VSMC Differential Expression (Dissection vs. Control)

library(ggpubr)
hdat_vsmc$VSMC_phenotype <- Idents(hdat_vsmc)
Idents(hdat_vsmc) <- "Disease"
de_VSMC <- FindMarkers(hdat_vsmc, ident.1 = "Dissection", ident.2 = "Control", verbose = FALSE, logfc.threshold = 0.25, min.pct = 0.25)
# Create a file of differentially expressed genes between VSMC1 and VSMC2
openxlsx::write.xlsx(de_VSMC, file = "../2_Output/Figure_2/VSMC_DEGs_Disease.xlsx", rowNames=T)
###
library(ggpubr)
plots_Disease_UP <- VlnPlot(hdat_vsmc, features = c("MACROD2", "CDK17", "FOS"), group.by = "Disease",
    pt.size = 0) & theme(axis.title.x = element_blank()) & scale_fill_manual(values = c("dodgerblue4", "goldenrod2"))
plots_Disease_DOWN <- VlnPlot(hdat_vsmc, features = c("PTPRQ", "HLA-B", "STAB1"), group.by = "Disease",
    pt.size = 0) & theme(axis.title.x = element_blank()) & scale_fill_manual(values = c("dodgerblue4", "goldenrod2"))
# ggarrange(plots_Disease_UP, plots_Disease_DOWN, ncol=1, nrow=2, common.legend = TRUE, legend="right")

pdf(file = "../2_Output/Figure_2/Dissection_up_Top.Violins.pdf", height = 3, width = 6)
plots_Disease_UP
dev.off()
pdf(file = "../2_Output/Figure_2/Dissection_down_Top.Violins.pdf", height = 3, width = 6)
plots_Disease_DOWN
dev.off()
##################
DoHeatmap(hdat_vsmc, features = rownames(de_VSMC), size = 3, disp.min = -2, disp.max = 2) + scale_fill_gradient2(low = "dodgerblue4", mid = "white", high = "coral2",midpoint = 0)

##################
pdf(file = "../2_Output/Figure_2/Dot.plot_top5markers.pdf", height = 10, width = 7)
Clustered_DotPlot(seurat_object = hdat_vsmc, features = rownames(de_VSMC), x_lab_rotate=F, k = 10)
dev.off()

#Volcano Plot
# Load packages
library(dplyr)
library(ggplot2)
library(ggrepel)
library(openxlsx)
# Read data from the web
options(ggrepel.max.overlaps = Inf)
results = mutate(de_VSMC, minuslogpvalue = -log(p_val), log2FC=avg_log2FC)
results<-results %>% filter(p_val!=0)
results$gene_name<-rownames(results)
results <- results %>% 
  mutate(., sig=ifelse(p_val<0.05 & log2FC>.5, 
                       "P < 0.05 and Fold-Change > 0.5", 
                       ifelse(p_val<0.05 & log2FC< 0-0.5,
                              "P < 0.05 and Fold-Change < -0.5", 
                              "Not Sig")
                       )
         )
results$sig<-factor(results$sig, 
levels = c("P < 0.05 and Fold-Change < -0.5",
  "Not Sig",
  "P < 0.05 and Fold-Change > 0.5")
  )
max(results$minuslogpvalue, na.rm = TRUE)
max(results$log2FC, na.rm = TRUE)
min(results$log2FC, na.rm = TRUE)
p = ggplot(results, aes(log2FC, minuslogpvalue)) + 
  theme_classic() +
  theme(axis.line = element_blank(),
        axis.ticks = element_blank()
  ) +
  geom_point(aes(fill=sig, size = minuslogpvalue),
             colour="black",
             shape=21,
             stroke = 0,
             alpha = 0.9) +
  geom_vline(xintercept=0.5, size=.5, linetype="dashed") +
  geom_vline(xintercept=-0.5, size=0.5, linetype="dashed") +
  geom_hline(yintercept=0-log(0.05), size=.5, linetype="dashed") +
  labs(x=expression(Log[2](Fold-Change)), y=expression(-Log[10](P-value))) + 
  xlim(min(results$log2FC, na.rm = TRUE),max(results$log2FC, na.rm = TRUE)) + 
  ylim(-0, max(results$minuslogpvalue, na.rm = TRUE)) + geom_hline(yintercept = 0, size = 1) + 
  geom_vline(xintercept=0, size=1) +
  scale_fill_manual(values=c("dodgerblue4", "darkgray", "goldenrod2")) +
  scale_size_continuous(range = c(.1, 3))

  p+
  geom_text_repel(data=top_n(filter(results, log2FC< -0.5), 20, minuslogpvalue), aes(label=gene_name)) +
  geom_text_repel(data=top_n(filter(results, log2FC>0.5), 20, minuslogpvalue), aes(label=gene_name)) +
  theme(text = element_text(size=20))

##
pdf(file = paste0("../2_Output/Figure_2/VSMC_Dissection.vs.Control_VolcanoPlot.pdf"), height = 5, width = 5)
  p+
  geom_text_repel(data=top_n(filter(results, log2FC< -0.5), 20, minuslogpvalue), aes(label=gene_name)) +
  geom_text_repel(data=top_n(filter(results, log2FC>0.5), 20, minuslogpvalue), aes(label=gene_name)) +
    theme(text = element_text(size=14), legend.position="none")
dev.off()
# 
# ##################
# DoHeatmap(hdat_vsmc, features = VSMC_type_DEs, group.by = "VSMC_phenotype",size = 3, disp.min = -2, disp.max = 2) + scale_fill_gradientn(colors = c("dodgerblue4", "white", "coral2"))
##################
pdf(file = "../2_Output/Figure_2/DotPlot_VSMC_1.v.2.pdf", height = 10, width = 5)
Clustered_DotPlot(seurat_object = hdat_vsmc, features = VSMC_type_DEs, group.by = "VSMC_phenotype", x_lab_rotate=F, k = 2)
dev.off()

Supplemental Table: R Session Information

All packages and setting are acquired using the following command:

sinfo<-devtools::session_info()
sinfo$platform
##  setting  value
##  version  R version 4.2.2 (2022-10-31)
##  os       macOS Big Sur ... 10.16
##  system   x86_64, darwin17.0
##  ui       X11
##  language (EN)
##  collate  en_US.UTF-8
##  ctype    en_US.UTF-8
##  tz       America/New_York
##  date     2023-01-14
##  pandoc   2.19.2 @ /Applications/RStudio.app/Contents/Resources/app/quarto/bin/tools/ (via rmarkdown)
sinfo$packages %>% kable( 
                         align="c", 
                         longtable=T, 
                         booktabs=T,
                         caption="Packages and Required Dependencies") %>% 
    kable_styling(latex_options=c("striped", "repeat_header", "condensed"))
Packages and Required Dependencies
package ondiskversion loadedversion path loadedpath attached is_base date source md5ok library
abind abind 1.4.5 1.4-5 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/abind /Library/Frameworks/R.framework/Versions/4.2/Resources/library/abind FALSE FALSE 2016-07-21 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
assertthat assertthat 0.2.1 0.2.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/assertthat /Library/Frameworks/R.framework/Versions/4.2/Resources/library/assertthat FALSE FALSE 2019-03-21 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
bslib bslib 0.4.2 0.4.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/bslib /Library/Frameworks/R.framework/Versions/4.2/Resources/library/bslib FALSE FALSE 2022-12-16 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
cachem cachem 1.0.6 1.0.6 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/cachem /Library/Frameworks/R.framework/Versions/4.2/Resources/library/cachem FALSE FALSE 2021-08-19 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
callr callr 3.7.3 3.7.3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/callr /Library/Frameworks/R.framework/Versions/4.2/Resources/library/callr FALSE FALSE 2022-11-02 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
cli cli 3.5.0 3.5.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/cli /Library/Frameworks/R.framework/Versions/4.2/Resources/library/cli FALSE FALSE 2022-12-20 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
cluster cluster 2.1.4 2.1.4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/cluster /Library/Frameworks/R.framework/Versions/4.2/Resources/library/cluster FALSE FALSE 2022-08-22 CRAN (R 4.2.2) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
codetools codetools 0.2.18 0.2-18 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/codetools /Library/Frameworks/R.framework/Versions/4.2/Resources/library/codetools FALSE FALSE 2020-11-04 CRAN (R 4.2.2) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
colorspace colorspace 2.0.3 2.0-3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/colorspace /Library/Frameworks/R.framework/Versions/4.2/Resources/library/colorspace FALSE FALSE 2022-02-21 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
cowplot cowplot 1.1.1 1.1.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/cowplot /Library/Frameworks/R.framework/Versions/4.2/Resources/library/cowplot FALSE FALSE 2020-12-30 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
crayon crayon 1.5.2 1.5.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/crayon /Library/Frameworks/R.framework/Versions/4.2/Resources/library/crayon FALSE FALSE 2022-09-29 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
data.table data.table 1.14.6 1.14.6 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/data.table /Library/Frameworks/R.framework/Versions/4.2/Resources/library/data.table FALSE FALSE 2022-11-16 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
DBI DBI 1.1.3 1.1.3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/DBI /Library/Frameworks/R.framework/Versions/4.2/Resources/library/DBI FALSE FALSE 2022-06-18 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
deldir deldir 1.0.6 1.0-6 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/deldir /Library/Frameworks/R.framework/Versions/4.2/Resources/library/deldir FALSE FALSE 2021-10-23 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
devtools devtools 2.4.5 2.4.5 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/devtools /Library/Frameworks/R.framework/Versions/4.2/Resources/library/devtools FALSE FALSE 2022-10-11 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
digest digest 0.6.31 0.6.31 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/digest /Library/Frameworks/R.framework/Versions/4.2/Resources/library/digest FALSE FALSE 2022-12-11 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
dplyr dplyr 1.0.10 1.0.10 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/dplyr /Library/Frameworks/R.framework/Versions/4.2/Resources/library/dplyr TRUE FALSE 2022-09-01 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
ellipsis ellipsis 0.3.2 0.3.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ellipsis /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ellipsis FALSE FALSE 2021-04-29 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
evaluate evaluate 0.19 0.19 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/evaluate /Library/Frameworks/R.framework/Versions/4.2/Resources/library/evaluate FALSE FALSE 2022-12-13 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
fansi fansi 1.0.3 1.0.3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/fansi /Library/Frameworks/R.framework/Versions/4.2/Resources/library/fansi FALSE FALSE 2022-03-24 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
farver farver 2.1.1 2.1.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/farver /Library/Frameworks/R.framework/Versions/4.2/Resources/library/farver FALSE FALSE 2022-07-06 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
fastmap fastmap 1.1.0 1.1.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/fastmap /Library/Frameworks/R.framework/Versions/4.2/Resources/library/fastmap FALSE FALSE 2021-01-25 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
fitdistrplus fitdistrplus 1.1.8 1.1-8 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/fitdistrplus /Library/Frameworks/R.framework/Versions/4.2/Resources/library/fitdistrplus FALSE FALSE 2022-03-10 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
fs fs 1.5.2 1.5.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/fs /Library/Frameworks/R.framework/Versions/4.2/Resources/library/fs FALSE FALSE 2021-12-08 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
future future 1.30.0 1.30.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/future /Library/Frameworks/R.framework/Versions/4.2/Resources/library/future FALSE FALSE 2022-12-16 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
future.apply future.apply 1.10.0 1.10.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/future.apply /Library/Frameworks/R.framework/Versions/4.2/Resources/library/future.apply FALSE FALSE 2022-11-05 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
generics generics 0.1.3 0.1.3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/generics /Library/Frameworks/R.framework/Versions/4.2/Resources/library/generics FALSE FALSE 2022-07-05 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
ggplot2 ggplot2 3.4.0 3.4.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ggplot2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ggplot2 TRUE FALSE 2022-11-04 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
ggrepel ggrepel 0.9.2 0.9.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ggrepel /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ggrepel FALSE FALSE 2022-11-06 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
ggridges ggridges 0.5.4 0.5.4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ggridges /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ggridges FALSE FALSE 2022-09-26 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
globals globals 0.16.2 0.16.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/globals /Library/Frameworks/R.framework/Versions/4.2/Resources/library/globals FALSE FALSE 2022-11-21 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
glue glue 1.6.2 1.6.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/glue /Library/Frameworks/R.framework/Versions/4.2/Resources/library/glue FALSE FALSE 2022-02-24 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
goftest goftest 1.2.3 1.2-3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/goftest /Library/Frameworks/R.framework/Versions/4.2/Resources/library/goftest FALSE FALSE 2021-10-07 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
gridExtra gridExtra 2.3 2.3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/gridExtra /Library/Frameworks/R.framework/Versions/4.2/Resources/library/gridExtra FALSE FALSE 2017-09-09 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
gtable gtable 0.3.1 0.3.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/gtable /Library/Frameworks/R.framework/Versions/4.2/Resources/library/gtable FALSE FALSE 2022-09-01 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
highr highr 0.10 0.10 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/highr /Library/Frameworks/R.framework/Versions/4.2/Resources/library/highr FALSE FALSE 2022-12-22 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
htmltools htmltools 0.5.4 0.5.4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/htmltools /Library/Frameworks/R.framework/Versions/4.2/Resources/library/htmltools FALSE FALSE 2022-12-07 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
htmlwidgets htmlwidgets 1.6.1 1.6.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/htmlwidgets /Library/Frameworks/R.framework/Versions/4.2/Resources/library/htmlwidgets FALSE FALSE 2023-01-07 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
httpuv httpuv 1.6.7 1.6.7 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/httpuv /Library/Frameworks/R.framework/Versions/4.2/Resources/library/httpuv FALSE FALSE 2022-12-14 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
httr httr 1.4.4 1.4.4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/httr /Library/Frameworks/R.framework/Versions/4.2/Resources/library/httr FALSE FALSE 2022-08-17 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
ica ica 1.0.3 1.0-3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ica /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ica FALSE FALSE 2022-07-08 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
igraph igraph 1.3.5 1.3.5 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/igraph /Library/Frameworks/R.framework/Versions/4.2/Resources/library/igraph FALSE FALSE 2022-09-22 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
irlba irlba 2.3.5.1 2.3.5.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/irlba /Library/Frameworks/R.framework/Versions/4.2/Resources/library/irlba FALSE FALSE 2022-10-03 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
jquerylib jquerylib 0.1.4 0.1.4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/jquerylib /Library/Frameworks/R.framework/Versions/4.2/Resources/library/jquerylib FALSE FALSE 2021-04-26 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
jsonlite jsonlite 1.8.4 1.8.4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/jsonlite /Library/Frameworks/R.framework/Versions/4.2/Resources/library/jsonlite FALSE FALSE 2022-12-06 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
kableExtra kableExtra 1.3.4 1.3.4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/kableExtra /Library/Frameworks/R.framework/Versions/4.2/Resources/library/kableExtra TRUE FALSE 2021-02-20 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
KernSmooth KernSmooth 2.23.20 2.23-20 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/KernSmooth /Library/Frameworks/R.framework/Versions/4.2/Resources/library/KernSmooth FALSE FALSE 2021-05-03 CRAN (R 4.2.2) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
knitr knitr 1.41 1.41 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/knitr /Library/Frameworks/R.framework/Versions/4.2/Resources/library/knitr TRUE FALSE 2022-11-18 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
labeling labeling 0.4.2 0.4.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/labeling /Library/Frameworks/R.framework/Versions/4.2/Resources/library/labeling FALSE FALSE 2020-10-20 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
later later 1.3.0 1.3.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/later /Library/Frameworks/R.framework/Versions/4.2/Resources/library/later FALSE FALSE 2021-08-18 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
lattice lattice 0.20.45 0.20-45 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/lattice /Library/Frameworks/R.framework/Versions/4.2/Resources/library/lattice FALSE FALSE 2021-09-22 CRAN (R 4.2.2) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
lazyeval lazyeval 0.2.2 0.2.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/lazyeval /Library/Frameworks/R.framework/Versions/4.2/Resources/library/lazyeval FALSE FALSE 2019-03-15 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
leiden leiden 0.4.3 0.4.3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/leiden /Library/Frameworks/R.framework/Versions/4.2/Resources/library/leiden FALSE FALSE 2022-09-10 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
lifecycle lifecycle 1.0.3 1.0.3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/lifecycle /Library/Frameworks/R.framework/Versions/4.2/Resources/library/lifecycle FALSE FALSE 2022-10-07 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
limma limma 3.54.0 3.54.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/limma /Library/Frameworks/R.framework/Versions/4.2/Resources/library/limma FALSE FALSE 2022-11-01 Bioconductor /Library/Frameworks/R.framework/Versions/4.2/Resources/library
listenv listenv 0.9.0 0.9.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/listenv /Library/Frameworks/R.framework/Versions/4.2/Resources/library/listenv FALSE FALSE 2022-12-16 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
lmtest lmtest 0.9.40 0.9-40 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/lmtest /Library/Frameworks/R.framework/Versions/4.2/Resources/library/lmtest FALSE FALSE 2022-03-21 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
magrittr magrittr 2.0.3 2.0.3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/magrittr /Library/Frameworks/R.framework/Versions/4.2/Resources/library/magrittr FALSE FALSE 2022-03-30 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
MASS MASS 7.3.58.1 7.3-58.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/MASS /Library/Frameworks/R.framework/Versions/4.2/Resources/library/MASS FALSE FALSE 2022-08-03 CRAN (R 4.2.2) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
Matrix Matrix 1.5.3 1.5-3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/Matrix /Library/Frameworks/R.framework/Versions/4.2/Resources/library/Matrix FALSE FALSE 2022-11-11 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
matrixStats matrixStats 0.63.0 0.63.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/matrixStats /Library/Frameworks/R.framework/Versions/4.2/Resources/library/matrixStats FALSE FALSE 2022-11-18 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
memoise memoise 2.0.1 2.0.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/memoise /Library/Frameworks/R.framework/Versions/4.2/Resources/library/memoise FALSE FALSE 2021-11-26 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
mime mime 0.12 0.12 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/mime /Library/Frameworks/R.framework/Versions/4.2/Resources/library/mime FALSE FALSE 2021-09-28 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
miniUI miniUI 0.1.1.1 0.1.1.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/miniUI /Library/Frameworks/R.framework/Versions/4.2/Resources/library/miniUI FALSE FALSE 2018-05-18 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
munsell munsell 0.5.0 0.5.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/munsell /Library/Frameworks/R.framework/Versions/4.2/Resources/library/munsell FALSE FALSE 2018-06-12 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
nlme nlme 3.1.161 3.1-161 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/nlme /Library/Frameworks/R.framework/Versions/4.2/Resources/library/nlme FALSE FALSE 2022-12-15 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
parallelly parallelly 1.33.0 1.33.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/parallelly /Library/Frameworks/R.framework/Versions/4.2/Resources/library/parallelly FALSE FALSE 2022-12-14 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
patchwork patchwork 1.1.2 1.1.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/patchwork /Library/Frameworks/R.framework/Versions/4.2/Resources/library/patchwork TRUE FALSE 2022-08-19 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
pbapply pbapply 1.6.0 1.6-0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/pbapply /Library/Frameworks/R.framework/Versions/4.2/Resources/library/pbapply FALSE FALSE 2022-11-16 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
pillar pillar 1.8.1 1.8.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/pillar /Library/Frameworks/R.framework/Versions/4.2/Resources/library/pillar FALSE FALSE 2022-08-19 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
pkgbuild pkgbuild 1.4.0 1.4.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/pkgbuild /Library/Frameworks/R.framework/Versions/4.2/Resources/library/pkgbuild FALSE FALSE 2022-11-27 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
pkgconfig pkgconfig 2.0.3 2.0.3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/pkgconfig /Library/Frameworks/R.framework/Versions/4.2/Resources/library/pkgconfig FALSE FALSE 2019-09-22 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
pkgload pkgload 1.3.2 1.3.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/pkgload /Library/Frameworks/R.framework/Versions/4.2/Resources/library/pkgload FALSE FALSE 2022-11-16 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
plotly plotly 4.10.1 4.10.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/plotly /Library/Frameworks/R.framework/Versions/4.2/Resources/library/plotly FALSE FALSE 2022-11-07 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
plyr plyr 1.8.8 1.8.8 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/plyr /Library/Frameworks/R.framework/Versions/4.2/Resources/library/plyr FALSE FALSE 2022-11-11 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
png png 0.1.8 0.1-8 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/png /Library/Frameworks/R.framework/Versions/4.2/Resources/library/png FALSE FALSE 2022-11-29 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
polyclip polyclip 1.10.4 1.10-4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/polyclip /Library/Frameworks/R.framework/Versions/4.2/Resources/library/polyclip FALSE FALSE 2022-10-20 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
prettyunits prettyunits 1.1.1 1.1.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/prettyunits /Library/Frameworks/R.framework/Versions/4.2/Resources/library/prettyunits FALSE FALSE 2020-01-24 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
processx processx 3.8.0 3.8.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/processx /Library/Frameworks/R.framework/Versions/4.2/Resources/library/processx FALSE FALSE 2022-10-26 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
profvis profvis 0.3.7 0.3.7 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/profvis /Library/Frameworks/R.framework/Versions/4.2/Resources/library/profvis FALSE FALSE 2020-11-02 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
progressr progressr 0.12.0 0.12.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/progressr /Library/Frameworks/R.framework/Versions/4.2/Resources/library/progressr FALSE FALSE 2022-12-13 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
promises promises 1.2.0.1 1.2.0.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/promises /Library/Frameworks/R.framework/Versions/4.2/Resources/library/promises FALSE FALSE 2021-02-11 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
ps ps 1.7.2 1.7.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ps /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ps FALSE FALSE 2022-10-26 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
purrr purrr 1.0.0 1.0.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/purrr /Library/Frameworks/R.framework/Versions/4.2/Resources/library/purrr FALSE FALSE 2022-12-20 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
R6 R6 2.5.1 2.5.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/R6 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/R6 FALSE FALSE 2021-08-19 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
RANN RANN 2.6.1 2.6.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/RANN /Library/Frameworks/R.framework/Versions/4.2/Resources/library/RANN FALSE FALSE 2019-01-08 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
RColorBrewer RColorBrewer 1.1.3 1.1-3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/RColorBrewer /Library/Frameworks/R.framework/Versions/4.2/Resources/library/RColorBrewer FALSE FALSE 2022-04-03 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
Rcpp Rcpp 1.0.9 1.0.9 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/Rcpp /Library/Frameworks/R.framework/Versions/4.2/Resources/library/Rcpp FALSE FALSE 2022-07-08 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
RcppAnnoy RcppAnnoy 0.0.20 0.0.20 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/RcppAnnoy /Library/Frameworks/R.framework/Versions/4.2/Resources/library/RcppAnnoy FALSE FALSE 2022-10-27 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
remotes remotes 2.4.2 2.4.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/remotes /Library/Frameworks/R.framework/Versions/4.2/Resources/library/remotes FALSE FALSE 2021-11-30 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
reshape2 reshape2 1.4.4 1.4.4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/reshape2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/reshape2 FALSE FALSE 2020-04-09 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
reticulate reticulate 1.27 1.27 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/reticulate /Library/Frameworks/R.framework/Versions/4.2/Resources/library/reticulate FALSE FALSE 2023-01-07 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
rlang rlang 1.0.6 1.0.6 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/rlang /Library/Frameworks/R.framework/Versions/4.2/Resources/library/rlang FALSE FALSE 2022-09-24 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
rmarkdown rmarkdown 2.19 2.19 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/rmarkdown /Library/Frameworks/R.framework/Versions/4.2/Resources/library/rmarkdown FALSE FALSE 2022-12-15 CRAN (R 4.2.2) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
ROCR ROCR 1.0.11 1.0-11 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ROCR /Library/Frameworks/R.framework/Versions/4.2/Resources/library/ROCR FALSE FALSE 2020-05-02 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
rstudioapi rstudioapi 0.14 0.14 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/rstudioapi /Library/Frameworks/R.framework/Versions/4.2/Resources/library/rstudioapi FALSE FALSE 2022-08-22 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
Rtsne Rtsne 0.16 0.16 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/Rtsne /Library/Frameworks/R.framework/Versions/4.2/Resources/library/Rtsne FALSE FALSE 2022-04-17 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
rvest rvest 1.0.3 1.0.3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/rvest /Library/Frameworks/R.framework/Versions/4.2/Resources/library/rvest FALSE FALSE 2022-08-19 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
sass sass 0.4.4 0.4.4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/sass /Library/Frameworks/R.framework/Versions/4.2/Resources/library/sass FALSE FALSE 2022-11-24 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
scales scales 1.2.1 1.2.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/scales /Library/Frameworks/R.framework/Versions/4.2/Resources/library/scales FALSE FALSE 2022-08-20 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
scattermore scattermore 0.8 0.8 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/scattermore /Library/Frameworks/R.framework/Versions/4.2/Resources/library/scattermore FALSE FALSE 2022-02-14 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
sctransform sctransform 0.3.5 0.3.5 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/sctransform /Library/Frameworks/R.framework/Versions/4.2/Resources/library/sctransform FALSE FALSE 2022-09-21 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
sessioninfo sessioninfo 1.2.2 1.2.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/sessioninfo /Library/Frameworks/R.framework/Versions/4.2/Resources/library/sessioninfo FALSE FALSE 2021-12-06 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
Seurat Seurat 4.3.0 4.3.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/Seurat /Library/Frameworks/R.framework/Versions/4.2/Resources/library/Seurat TRUE FALSE 2022-11-18 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
SeuratObject SeuratObject 4.1.3 4.1.3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/SeuratObject /Library/Frameworks/R.framework/Versions/4.2/Resources/library/SeuratObject TRUE FALSE 2022-11-07 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
shiny shiny 1.7.4 1.7.4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/shiny /Library/Frameworks/R.framework/Versions/4.2/Resources/library/shiny FALSE FALSE 2022-12-15 CRAN (R 4.2.2) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
sp sp 1.5.1 1.5-1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/sp /Library/Frameworks/R.framework/Versions/4.2/Resources/library/sp FALSE FALSE 2022-11-07 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
spatstat.data spatstat.data 3.0.0 3.0-0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/spatstat.data /Library/Frameworks/R.framework/Versions/4.2/Resources/library/spatstat.data FALSE FALSE 2022-10-21 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
spatstat.explore spatstat.explore 3.0.5 3.0-5 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/spatstat.explore /Library/Frameworks/R.framework/Versions/4.2/Resources/library/spatstat.explore FALSE FALSE 2022-11-10 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
spatstat.geom spatstat.geom 3.0.3 3.0-3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/spatstat.geom /Library/Frameworks/R.framework/Versions/4.2/Resources/library/spatstat.geom FALSE FALSE 2022-10-25 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
spatstat.random spatstat.random 3.0.1 3.0-1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/spatstat.random /Library/Frameworks/R.framework/Versions/4.2/Resources/library/spatstat.random FALSE FALSE 2022-11-03 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
spatstat.sparse spatstat.sparse 3.0.0 3.0-0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/spatstat.sparse /Library/Frameworks/R.framework/Versions/4.2/Resources/library/spatstat.sparse FALSE FALSE 2022-10-21 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
spatstat.utils spatstat.utils 3.0.1 3.0-1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/spatstat.utils /Library/Frameworks/R.framework/Versions/4.2/Resources/library/spatstat.utils FALSE FALSE 2022-10-19 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
stringi stringi 1.7.8 1.7.8 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/stringi /Library/Frameworks/R.framework/Versions/4.2/Resources/library/stringi FALSE FALSE 2022-07-11 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
stringr stringr 1.5.0 1.5.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/stringr /Library/Frameworks/R.framework/Versions/4.2/Resources/library/stringr FALSE FALSE 2022-12-02 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
survival survival 3.4.0 3.4-0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/survival /Library/Frameworks/R.framework/Versions/4.2/Resources/library/survival FALSE FALSE 2022-08-09 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
svglite svglite 2.1.0 2.1.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/svglite /Library/Frameworks/R.framework/Versions/4.2/Resources/library/svglite FALSE FALSE 2022-02-03 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
systemfonts systemfonts 1.0.4 1.0.4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/systemfonts /Library/Frameworks/R.framework/Versions/4.2/Resources/library/systemfonts FALSE FALSE 2022-02-11 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
tensor tensor 1.5 1.5 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/tensor /Library/Frameworks/R.framework/Versions/4.2/Resources/library/tensor FALSE FALSE 2012-05-05 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
tibble tibble 3.1.8 3.1.8 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/tibble /Library/Frameworks/R.framework/Versions/4.2/Resources/library/tibble FALSE FALSE 2022-07-22 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
tidyr tidyr 1.2.1 1.2.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/tidyr /Library/Frameworks/R.framework/Versions/4.2/Resources/library/tidyr FALSE FALSE 2022-09-08 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
tidyselect tidyselect 1.2.0 1.2.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/tidyselect /Library/Frameworks/R.framework/Versions/4.2/Resources/library/tidyselect FALSE FALSE 2022-10-10 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
urlchecker urlchecker 1.0.1 1.0.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/urlchecker /Library/Frameworks/R.framework/Versions/4.2/Resources/library/urlchecker FALSE FALSE 2021-11-30 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
usethis usethis 2.1.6 2.1.6 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/usethis /Library/Frameworks/R.framework/Versions/4.2/Resources/library/usethis FALSE FALSE 2022-05-25 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
utf8 utf8 1.2.2 1.2.2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/utf8 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/utf8 FALSE FALSE 2021-07-24 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
uwot uwot 0.1.14 0.1.14 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/uwot /Library/Frameworks/R.framework/Versions/4.2/Resources/library/uwot FALSE FALSE 2022-08-22 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
vctrs vctrs 0.5.1 0.5.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/vctrs /Library/Frameworks/R.framework/Versions/4.2/Resources/library/vctrs FALSE FALSE 2022-11-16 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
viridisLite viridisLite 0.4.1 0.4.1 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/viridisLite /Library/Frameworks/R.framework/Versions/4.2/Resources/library/viridisLite FALSE FALSE 2022-08-22 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
webshot webshot 0.5.4 0.5.4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/webshot /Library/Frameworks/R.framework/Versions/4.2/Resources/library/webshot FALSE FALSE 2022-09-26 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
withr withr 2.5.0 2.5.0 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/withr /Library/Frameworks/R.framework/Versions/4.2/Resources/library/withr FALSE FALSE 2022-03-03 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
xfun xfun 0.36 0.36 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/xfun /Library/Frameworks/R.framework/Versions/4.2/Resources/library/xfun FALSE FALSE 2022-12-21 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
xml2 xml2 1.3.3 1.3.3 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/xml2 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/xml2 FALSE FALSE 2021-11-30 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
xtable xtable 1.8.4 1.8-4 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/xtable /Library/Frameworks/R.framework/Versions/4.2/Resources/library/xtable FALSE FALSE 2019-04-21 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
yaml yaml 2.3.6 2.3.6 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/yaml /Library/Frameworks/R.framework/Versions/4.2/Resources/library/yaml FALSE FALSE 2022-10-18 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library
zoo zoo 1.8.11 1.8-11 /Library/Frameworks/R.framework/Versions/4.2/Resources/library/zoo /Library/Frameworks/R.framework/Versions/4.2/Resources/library/zoo FALSE FALSE 2022-09-17 CRAN (R 4.2.0) /Library/Frameworks/R.framework/Versions/4.2/Resources/library